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Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, Massachusetts, approved on December 31, 2020 (review received on June 22, 2020)

Obviously, our immune system can significantly inhibit the growth of cancer cells. This process is called "cancer immunoediting." Based on this model, if programmed cell death (apoptosis) mediated through the immune surveillance process is unsuccessful, tumor cells may enter the equilibrium phase, at which stage they are either immunologically maintained or “edited”, resulting in A large number of tumor mutations appear in the escape stage. Microbiological factors have been found to affect cancer immune editing. In this study, we identified a core player in these processes, called the major vault protein (MVP). The combination of certain bacterial signal molecules and MVP strongly regulates apoptosis signals, which inspires new ideas and approaches to understand the relationship between humans and bacteria and fight cancer.

Major vault protein (MVP) mediates a variety of cellular responses, including the resistance of cancer cells to chemotherapy and the protection of Pseudomonas aeruginosa inflammation. Here, we report the use of light-sensitive probes to identify MVP as the target of N-(3-oxo-dodecanoyl) homoserine lactone (C12), which is a certain type including Pseudomonas aeruginosa Quorum sensing signal of Proteobacteria. Treatment of normal cells and cancer cells with C12 or other N-acyl homoserine lactones (AHL) leads to rapid translocation of MVP into lipid raft (LR) membrane components. Like AHL, inflammatory stimulation also induces LR localization of MVP, but C12 stimulation reprograms (functionalizes) the biological activity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 to the LR. These functionalized membranes control the signal transduction process induced by AHL because MVP regulates the protein kinase p38 pathway to reduce programmed cell death. Since MVP is the structural core of large particles called fornix, our research results indicate that MVP fornix acts as a sentinel mechanism that can fine-tune the inflammatory activation process, such as by immune surveillance cytokines (including tumor necrosis factor-related apoptosis-inducing ligands) )-Mediated apoptosis signal transduction. trace).

Cancer immunoediting is a dynamic process consisting of three stages-elimination, balance and escape (1, 2). According to this hypothesis, if immune surveillance effectors cannot mediate apoptosis in new tumors, transformed cells will enter the equilibrium phase, where they are either maintained for a long time or immune "edited" to produce new tumors during the escape phase. Variation group (2). The inflammatory microenvironment-including microbial metabolites such as lipopolysaccharide (LPS) and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-affects those undergoing any of the three stages (2⇓ ⇓ ⇓-6) of cancer immunoediting The signal transduction process of cells, and can promote the production of tumor cells resistant to pro-apoptotic immune surveillance factors, such as TNF-related apoptosis-inducing ligand (TRAIL) (7⇓-9). It is worth noting that the resistance of tumor cells to TRAIL-induced apoptosis is overcome in the presence of gram-negative bacteria that produce N-acyl homoserine lactones (AHL), such as C12 (10). The ability of AHLs to affect pro-apoptotic signals in normal and transformed cells (10, 11) suggests a mechanism by which the immune editing process is reprogrammed in response to protein bacterial metabolites.

Previously, we developed a series of C12 dual-function photosensitive probes for chemical proteomics analysis (SI appendix, Figure S1, compounds 6, 7 and 8), of which P6 is expressed in normal human bronchial epithelial (NHBE) cells The same agonistic properties as C12 (12). In order to identify the specific targets of C12 in human cells, we performed amino acid (SILAC) experiments on NHBE cells treated with P6 or a control compound (carrier or C12), and tested them with ultraviolet light ( UV) irradiated with covalent labeling followed by stable isotope labeling, and then through cleavage, orthogonal conjugation with biotin azide and affinity purification, the obtained proteome samples were analyzed by liquid chromatography tandem mass spectrometry (Figure 1A). Among the several P6 targeting proteins identified in these studies, the major vault protein (MVP) consistently showed a high labeling rate compared to the control (Figure 1 B and C and SI appendix, Figure S2; all identified using this method) The proteins are listed in data set S1).

Chemical proteomics analysis identified MVP as a potential target for C12. (A) General scheme of chemical proteomics strategy, using probe P6 (left, bottom). (B) Representative LC-MS spectrum of identified MVP peptides. The SILAC ratio and peptide sequence are shown at the top. (C) A representative example of the competitive effect of C12 on P6 mediated MVP markers. Compared with the probe labeling alone (blue), there is less probe labeling in the presence of the natural molecule C12 (red). (D) The image on the left shows the barrel structure of the vault. The MVP unit is marked in green; the top and bottom arrows mark the vault and shoulder areas, respectively. The figure on the right shows the computer docking simulation results of the MVP-C12 interaction, showing that C12 (white) is positioned close to the arch shoulder area (yellow). (E) A representative example of the identification of P6 peptide adducts from a vault labeling experiment using a hydrazine (N2H4) cleavable levulinic acid linker. The m/z values ​​of matched light (14N) and heavy (15N) adduct pairs that recognize MVP peptides are shown, producing characteristic bimodal signals. The images and molecules shown in A, D, and E are not drawn to scale. For other information related to the above experiment, please refer to the SI appendix, figure. S2-S4.

The evolutionarily conserved protein MVP is expressed as large barrel-shaped particles called fornix, which are found in eukaryotes, including human cells (Figure 1D) (13⇓ ⇓ –16). The heterologous expression of MVP leads to the assembly of arched particles, which are structurally indistinguishable from their naturally occurring counterparts (16⇓ –18). Therefore, to validate our cell-based proteomics findings, we checked whether our C12-based probe P6 targets the recombinant human library. As expected, P6 marked the reorganization vault in a dose-dependent manner (SI appendix, Figure S3A). Importantly, in the presence of C12, the P6 label of MVP was significantly reduced (SI appendix, Figure S3B), indicating that these two compounds interacted with the vault in a similar manner. To further confirm these findings, the recombinant library P6 adduct was additionally coupled with a biotinylated azide containing a levulinic acid-based cleavable linker. After streptavidin-based affinity purification, the linker was cleaved with an equimolar mixture of 15N- and 14N-hydrazine to release the P6 labeled peptide, resulting in the characteristic bimodal distribution observed in subsequent mass spectrometry analysis (Figure 1E). According to the dome crystal structure (19), the identified P6 peptide adduct (SI appendix, Figure S4) is located in the shoulder (residues 445 to 461) and cap (residues 724 to 734) regions of MVP. Computer docking simulation of the C12 dome complex consistent with ours (Figure 1D, panel on the right). Interestingly, these results indicate that C12 is located near the MVP domain, which may also be involved in the interaction with lipid rafts (LR) (16, 19).

Previous studies on human airway epithelial cells have shown that MVP is recruited to LRs in response to Pseudomonas aeruginosa. The activation of MVP translocation seems to depend on the integrity of LPS, although the role of other bacterial metabolites in this cellular process is still not significant. Clear (20). To evaluate whether C12 also needs to induce LR-localized MVP (rMVP), we compared the subcellular localization of MVP in NHBE cells treated with wild-type Pseudomonas aeruginosa or isogenic mutant strains lacking lasI, which is responsible for C12 (21) Synthesis. The response of rMVP to wild-type bacteria increased significantly; however, we found that when cells were treated with C12-deficient bacteria, rMVP was significantly reduced (Figure 2A). It is worth noting that the residual levels of rMVP observed in response to C12-deficient Pseudomonas aeruginosa may be due to the activity of other bacterial stimuli, such as LPS (20) or its basic core component Kdo2-lipid A (KLA) (SI appendix, Figure S5).

Pseudomonas aeruginosa that produces C12 and C12 alone induce the translocation of MVP to the LR fraction. (A) Comparison of the reactivity of NHBE cells to Pseudomonas aeruginosa wild-type (wt) or lasI mutant (ΔlasI). MVP was expressed by WB analysis using cell fractions containing cytosol (C) or LRs , Vimentin (marker of LR fraction), and HSP70 (marker of cytoplasmic fraction). (B) NHBE cells were stimulated with C12 or its unnatural R-stereoisomer (C12R), and cell samples were analyzed such as A and PARP4 expression. (C, D) A549 lung cancer cell line (C) and normal human CBM (D) were stimulated with C12 and analyzed as shown in B. For additional information related to the above experiments, please refer to the SI appendix, Figures S5 and S6.

In order to clarify the specific role of C12 in the recruitment of MVP to the LR fraction, we examined the induction of rMVP in NHBE cells treated with the natural S- or unnatural R-stereoisomer of C12 (SI appendix, Figure S6 A and B ). The subcellular localization of type 4 poly(ADP-ribose) polymerase (PARP4), which is a component of the fornix (22), was also tested. For the C12 response with natural stereochemistry, similar LR localization patterns were observed for the two components of the dome, while the non-natural enantiomer was completely inactive (Figure 2B). The induction of LR-localized vault components was not limited to NHBE cells, but was also observed in human lung cancer cell line A549 (Figure 2C) treated with C12 and normal human umbilical cord blood-derived macrophages (Figure 2D). The lipophilicity of C12 and other long-chain AHLs allows direct interaction with the plasma membrane (23, 24), therefore, the observed fornix components are recruited into lipid-rich membrane rafts in response to AHL (SI appendix, Figure S6 CE) It is consistent with the assumption that mammalian perception of AHL occurs in the membrane positioning system (23).

The observation that C12 activates apoptotic signaling in various cell types and MVP regulates the resistance of cancer cells to pro-apoptotic anti-cancer agents (13, 25) prompted us to study the biochemical characteristics related to the apoptotic effect of C12 and its effects. The ability to induce rMVP in lung cancer cell line A549. Western blot (WB) analysis showed that C12-induced apoptotic caspase activation and the kinetics of fornix component recruitment to the LR part are similar; it is worth noting that only caspase- 8 (casp8) is transferred to the LR fraction in response to C12 (Figure 3A), which is very similar to the activation of this initial caspase in cancer cells that respond to TRAIL (26). Interestingly, in wild-type and MVP-deficient bone marrow-derived macrophages (BMDM), C12 also induced the recruitment of casp8 to the LR part (Figure 3B), indicating that the observed translocation of fornix components and casp8 are two An independent response to AHL-mediated events.

MVP attenuates the C12-induced apoptosis signal. (A) Stimulate A549 cells with C12, prepare subcellular fractions and analyze the expression of fornix fraction, apoptotic caspase and loading control markers by WB, as shown. (B) BMDM from wild-type (MVP+/+) and MVP-deficient (MVP-/-) mice was treated with C12 as shown in the figure, and the expression of subcellular parts (cytoplasm [C] and LR) was analyzed by WB casp8, Vault components (MVP and PARP4) and vimentin. (C, D) WB analysis monitors the cleavage of PARP and casp3 and the phosphorylation of eIF2α (p-eIF2α) in extracts from wild-type and MVP-deficient MEF (C) or BMDM (D), as shown in the figure. (E) Overview of casp8, casp9 or casp3 activity in BMDM treated with C12. (F) WB analysis monitors the activation of the initial caspases (casp8 and casp9) from the C12-treated MVP+/+ or MVP-/- BMDM extract, as shown in the figure. For other information related to the above experiment, please refer to the SI appendix, figure. S7 and S8.

To clarify whether MVP is involved in the regulation of C12-induced apoptosis signals, we used cells from wild-type and MVP knockout mice. Titration experiments showed that compared with wild-type counterparts, the viability of BMDM and mouse embryonic fibroblasts (MEF) derived from MVP-deficient mice was significantly reduced in the presence of C12 (SI Appendix, Figure S7A), indicating that MVP has Helps prevent cell death induced by C12. We also observed that with their wild-type counterparts (Figure 3 C and D and SI appendix, Figure S7B). The MVP dependence of the C12-mediated response is specific to apoptotic signaling because of phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) and splicing of XbpI messenger RNA (mRNA), which is the adaptive endoplasmic reticulum The (ER) indicator marks the stress pathway, showing similar patterns in C12 activation, MVP deficiency, and wild-type cells (Figure 3C and D and SI appendix, Figure S7C).

Apoptosis is initiated through two different signaling pathways: an internal pathway triggered by the effector caspase-9 (casp9) and an external pathway that depends on the effector casp8 (27). Although C12 activates the two effector caspases (Figure 3A), the MVP dependence of this response still needs to be addressed to explain the observed difference in sensitivity of MVP-deficient cells and wild-type cells to C12. We found that lack of MVP significantly increased the C12-induced casp8 activity, while the induction of casp9 was not related to MVP (Figure 3E and F). Other AHLs revealed similar MVP-dependent activation of apoptosis signaling (SI Appendix, Figure S8A). In additional experiments, inflammatory stimuli, such as LPS and KLA, were also tested as controls. Consistent with the ability of inflammatory stimuli to induce anti-apoptotic responses by activating the NF-κB pathway (6, 9), LPS and KLA do not induce apoptosis signaling in wild-type or MVP-deficient cells (SI Appendix, Figure S8B). Therefore, this set of findings indicates that the vault plays an important role in the protection against C12-induced apoptosis through the casp8-dependent mechanism. It can also explain the chemoresistance associated with the up-regulation of MVP in cancer cells (13, 25).

Like LPS, C12 activates the p38 protein kinase pathway, which negatively regulates apoptosis in LPS-activated cells (28). However, although LPS also induces inflammatory responses in macrophages by activating NF-κB signaling (6), C12 does not. In contrast, C12 inhibits LPS-mediated transcriptional induction of NF-κB target genes encoding TNF and other inflammation regulators, and exhibits anti-inflammatory activity independent of TLR4 (11). We found that lack of MVP had no effect on the induction of TNF and interleukin-1β (IL-1β) expression in LPS-activated macrophages (Figure 4A, cell line treated with LPS only). In addition, MVP seems to be dispensable for the anti-inflammatory activity of C12, because LPS-induced TNF production is strongly and equally inhibited by C12 in both wild-type and MVP-deficient BMDM (Figure 4A and B). However, although inflammatory stimuli such as LPS and TNF induced similar p38 phosphorylation (p-p38) profiles in wild-type and MVP-deficient cells (SI Appendix, Figure S9), C12-induced p-p38 was significant in MVP Impaired-lack of BMDM and MEF (Figure 4C). In contrast, C12-mediated effects on the phosphorylation status of other MAPKs, such as JNK and ERK, are similar in wild-type and MVP-deficient macrophages (see Figure 3 for p-JNK and p-ERK1/ 2 WB results). 4C). At present, it is not clear whether MVP is directly involved in C12-induced p38 activation, or whether MVP/fornix as a signal component transporter (29, 30) affects the expression of p38 in the intracellular compartment. Interestingly, WB analysis of the expression of p38 and JNK in the cytoplasm and LR fractions showed that the cytoplasmic expression of these two proteins was not affected in MVP-deficient macrophages; however, although the expression of JNK is limited to the cytoplasm, P38 was also detected in the LR fraction, and the level of p38 expressed by LR in MVP-deficient cells was significantly reduced (Figure 4D). We also noticed that MVP and p38-deficient cells respond similarly to C12-induced apoptotic signaling (Figure 4 E and F; see also SI appendix, Figure S10), indicating the role of the MVP-p38 axis in cells. Survival in macrophages activated by stimuli other than C12 or a combination of different stimuli (30, 31).

The lack of MVP selectively affects the pro-survival activity of C12-induced p38 signaling. (A) WB analysis monitors the expression of TNF and interleukin 1β (IL-1β) in extracts stimulated with LPS in the presence of the specified dose of C12 in wild-type (WT) and MVP-deficient BMDM. (B) TNF secretion of WT or MVP-deficient (MVP ko) BMDM stimulated with LPS in the presence of the specified dose of C12. (C) WB analysis of p38, eIF2α, JNK, ERK1/2 and their phosphorylated forms, as well as MVP from WT and MVP-deficient BMDM extracts, as shown in the figure. (D) Analyzed the expression of p38, JNK and loading control (vim, abbreviation of vimentin) from three independent cytoplasmic and LR fractions of WT (1 to 3) or MVP-deficient BMDM (ko; 4 to 6) . (E) WB analysis of PARP, p-p38, p-eIF2α and actin in cell extracts of C12-stimulated WT and p38-deficient cells. (F) As shown in the figure, compare casp8 activity in WT and MVP or p38-deficient cells stimulated with C12 (10 mM). (G) After transfection with MVP-specific siRNA (MVP) or non-targeting control siRNA (NT), untransfected (control) and siRNA-transfected human CBMs were treated with C12 for a specified time, and cell extracts were analyzed by WB The MVP, p-p38, p-CREB, p-eIF2α and actin. The same group of CBM was also incubated with C12 for 24 hours, and cell viability was measured and displayed (bottom box; three biological replicates) as the percentage of survival (100%) of untreated cells.

The integrity of the NF-κB or p38 signaling pathway is essential for the protection of macrophages from apoptosis induced by pathogens or inflammatory stimuli (28, 32, 33). In TLR4-activated mouse macrophages, the survival activity of the IKKβ-NF-κB module and the phosphorylation-dependent activation of the p38-mediated transcription factor CREB ​​reduce the pro-apoptotic immune surveillance signal transduction (33). The addition of C12 to macrophages pretreated with TLR4 ligand and the observation of CREB phosphorylation (34) prompted us to examine whether C12 alone can activate CREB in human cord blood-derived macrophages (CBM). We observed that C12 induced strong phosphorylation of CREB​​, which was consistent with the activation of p38, but was damaged when CBM was pretreated with MVP targeting small interfering RNA (siRNA) (Figure 4G); in addition, the expression of MVP Reduction impairs the survival of CBM (Figure 4G, bottom). The MVP-p38 axis is involved in the production of macrophage scavenger receptor 1 (MSR1)-mediated immune surveillance cytokine TNF and the regulation of apoptosis signaling in the macrophage-like cell line RAW264.7 activated by fucoidan. This may be related to the previously reported observations, the ligand of MSR1 (29).

Death receptors, such as TNF receptor type 1 (TNFR1) and TRAIL receptor type 2 (also known as DR5), regulate the immune surveillance process through the activation of new cancer cell apoptosis induced by ligands (2⇓ ⇓ ⇓ ⇓ ⇓ ⇓ -9). Since the interaction between C12 and plasma membrane (23, 24) leads to TNF-independent activation of TNFR1-mediated apoptosis signaling (24), we examined TNFR1, TRADD (TNF receptor related death domain), DR5, C12 Or the apoptotic adaptor proteins FADD, caspase-8 and vault components in CBM simulated by TNF. Keeping in mind that MSR1 has been identified as a binding partner of MVP (29), these studies also included analysis of the location of MSR1. WB analysis of the subcellular fractions from C12 or TNF stimulated cells showed that the fornix components (MVP and PARP4) and MSR1 induced by stimulation were rapidly recruited into the LR fraction (Figure 5A). We also found that although low levels of LR-localized TNFR1 and DR5 were detected in response to TNF, C12 treatment significantly increased the recruitment of two death receptors to the LR part, and the presence of TRADD, FADD and caspase-8 was also obvious (Figure 5B). Interestingly, the similarity between the localization patterns of DR5 and MSR1 in the cytoplasm can be repeatedly observed at later time points after C12 stimulation (Figure 5A and B), which may be related to the C12-induced ER stress response (35) and Independent cell apoptosis mediated by TRAIL-DR5 (36).

Recruiting MVP to the LR platform can regulate immune editing and immune surveillance processes. (A) WB analysis monitors the expression of fornix components (MVP and PARP4), MSR1 and vimentin in CBM subfractions stimulated by C12 or TNF. (B) Additional analysis of the subtraction shown in A was performed to determine the expression of the specified protein. (C) Subfractions (C [cytoplasm], M [membrane], and LR) are prepared from CBM stimulated by LPS, and the expression of the specified protein is analyzed. (D) The relative level of radioactivity bound to sphingomyelin (SM) after treating 3H-Sph metabolically labeled macrophages with natural (C12S) or unnatural (C12R) enantiomers; samples from untreated cells Used as a control. For additional information, please refer to the SI appendix, Figure S9. (E and F) CBM was stimulated with the S- or R-enantiomer of C12, and the prepared subfraction (30 minutes after stimulation) was analyzed for the indicated expression on a set of proteins (E); at the same time, the total The expression of the p38 or eIF2α phosphorylated form of the cell extract was analyzed, and actin was used as a loading control (F). (G) After transfection with MVP-specific siRNA (MVP) or non-targeting control siRNA (NT), A549 cells were treated with C12 (5 μM), TRAIL (10 ng/mL) or a combination of the two stimuli for 3 hours And as shown, the cell extract was analyzed by WB for PARP and casp8 lysis and the full length of casp8 and actin. WB also verified the silencing of MVP expression in these cells (insert bottom). (H) In the presence of the specified dose of TRAIL, incubate the same group of A549 transfected cells with or without C12 (5 μM) for 18 hours, and evaluate the cell survival rate.

C12 is only produced in certain Gram-negative bacteria, which also have LPS as a key effector for inflammation and immune surveillance of macrophage responses. Therefore, we examined the subcellular localization of MSR1, fornix components, and death receptor signaling partners in CBM simulated by LPS. The analysis results shown in Figure 5C show that the fornix components (MVP and PARP4) and MSR1 are rapidly recruited to the LR in response to LPS, while the localization of casp8, death receptors (DR5 and TNFR1) and FADD are not affected, and is induced by LPS. The endogenous expression of TNF protein (TNF p26 and TNF p17) is consistent; the change of TNFR1 expression pattern is consistent with the recruitment of TRADD into the membrane fraction. Therefore, like C12 or TNF, cells respond to LPS by translocating the fornix component and MSR1 into the LR fraction, indicating that the LR recruitment of the MVP-MSR1 complex is the result of a common process triggered in the plasma membrane to respond These obviously different stimuli.

In the plasma membrane, LRs are a collection of cholesterol, sphingolipids and proteins. They can be transduced through lipid and/or protein-mediated signaling, such as lipid content/structure changes, multivalent ligand binding or protein oligomerization binding Together, the raft platform has biological activity in cell membrane function (37). For example, response to inflammatory mediators (such as LPS) and immune surveillance cytokines (such as TNF and TRAIL) trigger the conversion of sphingomyelin (SM) to ceramide (Cer), resulting in the formation of a Cer-rich raft platform that can regulate resistance and cell growth And the pro-apoptotic cell process necessary for death (38⇓ ⇓ ⇓ –42). It is worth noting that Cer induces cell death (43), but it can also be hydrolyzed into fatty acids and sphingosine (Sph). Then, through the sphingolipid salvage pathway (42), the acylation of Sph recycles Cer, which can further Metabolism to SM; therefore, the homeostasis of lipids in the plasma membrane and the anti-apoptotic effect of NF-κB signaling promote the survival of cells activated by LPS or TNF (42, 44). On the other hand, C12 inhibits TNF-mediated activation of NF-κB signaling (34), but activates apoptosis through TNFR1 oligomerization (24) and is consistent with the recruitment of TNFR1/DR5, its apoptotic linker, and casp8 to LRs (Figure 5B). These findings indicate that the lipophilic interaction of C12 with the plasma membrane promotes the formation of Cer-rich raft platforms, but a similar interaction with the ER membrane (the occurrence of Cer de novo synthesis (45)) may affect sphingolipids Ways to remedy. Although further research is needed to determine the mechanism, our 3H-Sph metabolic labeling experiments on macrophages show that the addition of C12 significantly weakens the incorporation of 3H-Sph into Cer, especially SM (Figure 5D and SI appendix, Figure S11A-C ). These findings indicate that the interaction of C12 with the plasma membrane disrupts the metabolically balanced sphingolipid component, thereby triggering protein-mediated signal transduction activation. It is worth noting that the natural stereochemistry of C12 is to regulate sphingolipid metabolism (SI appendix, Figure S11B) and to recruit death receptors containing proteins and the apoptotic mechanism of casp8 to the LR platform, and to mediate p38 and ER stress induced by C12. Induced phosphorylation of eIF2α (Figure 5 E and F). Importantly, consistent with our previous observations on NHBE cells, the translocation of dome components (MVP and PARP4) to the LR fraction was also induced in CBM, stimulated only by the natural S-enantiomer of C12 ( SI appendix, Figure S11D), further supports the specific recognition of C12 by the MVP vault.

Elevated MVP expression in cancer cells is associated with resistance to anti-cancer treatments (30, 46), indicating the role of Vault in the process of immune editing/immune surveillance. It is worth noting that lung cancer A549 cells express high levels of MVP (46) and show resistance to apoptosis induced by the immune surveillance cytokine TRAIL (7), but C12 restores the sensitivity of tumor cells to TRAIL (10) . The effect of C12 on the anti-cancer activity of TRAIL may be related to the destruction of sphingolipid metabolism, because the apoptotic response to TRAIL+C12 is blocked by the Cer metabolism inhibitor D-MAPP (47), while bortezomib-mediated effects TRAIL-induced apoptosis is not affected by D-MAPP (SI Appendix, Figure S11E); in addition, D-MAPP blocks C12-induced signal transduction, while the response to LPS is not affected (SI Appendix, Figure S11 F and G). The MVP library has been identified as an anti-apoptotic effector of C12, and we examined whether MVP is related to the resistance of A549 cells to TRAIL. As expected, the apoptotic signal in A549 cells transfected with control siRNA was slightly increased under the combination of TRAIL and C12, while the decrease in MVP expression mediated by siRNA resulted in a significant increase in the apoptotic signal (Figure 5G). Titration experiments confirmed that MVP-specific siRNA targeting cells are more sensitive to TRAIL (Figure 5H). Therefore, the observed involvement of MVP in TRAIL-induced apoptosis of lung cancer cells may indeed regulate the cancer immune editing/immune surveillance process, especially during Pseudomonas aeruginosa infection.

Our results show that the MVP vault has a specific function in recognizing C12 and other AHLs, as an indicator of the presence of AHL-producing proteobacteria (such as Pseudomonas aeruginosa). By cooperating with the TLR4-dependent response of LPS (3, 6, 9, 28), the fornix can promote the survival of activated cells, thereby coupling the process of host immune surveillance (2, 9, 10) (SI appendix, Figure 2). S11C) and pathogen elimination (20, 48) to solve the cell's response to classic inflammatory stimuli (including TNF). Therefore, it is necessary to further study the structure-activity relationship between Vault components to understand their therapeutic potential for inflammation-mediated pathologies (including cancer).

All chemical reagents used in organic synthesis were purchased from Sigma-Aldrich or Acros (Thermo Fisher Scientific) and can be used without further purification. The solvent is dried with Mbraun Solvent Purification System. The reaction was monitored by thin layer chromatography (TLC) using commercially available glass plates pre-coated with silica (0.25 mm, Merck 60 F254) and developed with potassium permanganate. Flash chromatography is performed on Merck 40 to 63 μm silica gel. Use Bruker Avance DPX400 or DMX500 for NMR analysis. The spectrum is calibrated based on the residual solvent signal. All AHLs, including C12 and C12R, are synthesized according to the published procedure (49⇓ -51). The synthesis of P6 has been described previously (52). The purity of all compounds was confirmed by liquid chromatography-mass spectrometry (LC-MS) and NMR analysis. In addition, the quantitative QCL-1000 color-developing limulus amebocyte lysate assay (BioWhittaker, Inc.) proved that C12 and other AHL preparations are endotoxin-free. [3-3H]-D-erythrosphingosine (20 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. Sphingolipids (Cer, Sph, SP1, DMS and other lipids) were purchased from Matreaya. Generally, synthetic molecules are dissolved in dimethyl sulfoxide (DMSO) or ethanol at 200 times the required concentration, and aliquots are stored at -20 °C. LPS (E. coli serotype 055:B5) was purchased from Sigma-Aldrich, and the preparation method of Salmonella Minnesota Re595 LPS was as previously described (53). The core structural component of LPS, also known as Di[3-deoxy-D-manno-octulosonyl]-lipid A, Kdo2-Lipid A or KLA, was obtained from Avanti. The supernatant level of TNF or IL-6 in the sample was measured by enzyme-linked immunosorbent assay (BD Biosciences Pharmingen).

The cells are plated at 20% confluence in the culture medium and grown to approximately 90% confluence. The stock solution of probe and C12 was dissolved in DMSO. Change the medium 12 hours before the experiment. The compound is added to the concentration indicated in the legend and the cells are incubated for 30 minutes. The cells were washed once with cold phosphate buffered saline (PBS) and irradiated on a UV stage at 4°C with a wavelength of 365 nm for 15 minutes. Use a cell scraper to collect the cells and store at -80°C. For the SILAC experiment, labeling was performed in a similar manner, but the cells were grown in SILAC Dulbecco's modified Eagle medium (DMEM) and dialyzed fetal bovine serum (FBS), purchased from Biological Industries, supplemented with isotopically-rich arginine and Lysine was used as a heavy medium, or had the same light amino acids, and was purchased from Sigma-Aldrich.

CuAAC chemistry was performed according to the protocol of Cravatt Laboratories (54). The protein concentration of each sample was measured using the bicinchoninic acid BCA protein assay (Pierce BCA protein assay kit; Thermo Fisher Scientific), and normalized by diluting with PBS to approximately 1 to 3 mg/mL. For each sample, take 1 mL of the proteome and add the following reagents: Azidorhodamine (10 µL 10 mM in DMSO, final concentration: 100 µM); Tris(2-carboxyethyl)phosphine (TCEP; 20 µL) 50 mM PBS, freshly prepared, final concentration: 1 mM); Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (60 µL 1.7 mM in 1:4 DMSO: t-butanol, final concentration: 100 µM); CuSO4 (20 µL 50 mM double distilled water [DDW], final concentration: 2 mM). The mixture was allowed to react on a shaker at room temperature for 1 hour. Finally, the samples were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gel, and the ImageQuant LAS 4000 imager (Fujifilm) set to Cy3 fluorescence mode (excitation/emission = 520 nm/575 nm) ) Observe the gel.

The sample preparation was carried out according to the protocol of Cravatt Laboratories (54). For the SILAC sample, the heavy protein group and the light protein group are combined at a ratio of 1:1 before continuing. CuAAC chemistry was performed as described above, using biotin azide instead of rhodamine azide. Use methanol/chloroform (MeOH/CHCl3) precipitation to remove excess reagent: transfer the sample to a 15 mL conical tube on ice, add 2 mL of cold MeOH, add 0.5 mL of cold CHCl3, and vortex the sample. Next, add 1.0 mL of cold PBS, vortex the sample and centrifuge at 5,000 rpm for 10 minutes. Remove the top and bottom layers, leaving the protein interface, and wash the sample with 1:1 MeOH:CHCl3 (1.0 mL, 3x). Add 2 mL MeOH, sonicate the sample with a probe sonicator, and then add 0.5 mL CHCl3. Finally, the sample was centrifuged at 5,000 rpm for 10 minutes to precipitate the protein and remove the supernatant. Next, the sample is denatured and reduced: 500 µL of 6M urea (in PBS, freshly prepared) and 50 µL of pre-mixed 100 mM TCEP/300 mM K2CO3 solution (in PBS) are added, then the sample is sonicated and subjected to 37 Incubate at °C for 30 minutes in a shaker. Next, add 70 µL of 400 mM iodoacetic acid and incubate at room temperature for 30 minutes in the dark, add 140 µL of 10% SDS in PBS, and dilute the sample with 5.5 mL of PBS.

Enrichment and trypsinization were performed according to the protocol of Cravatt Laboratories (54). The enrichment is carried out as follows: add the sample to 100 µL streptavidin beads (the beads are washed 3 times with 500 µL PBS), and incubate on a shaker for 1.5 hours at room temperature. Centrifuge the sample to pellet the beads, and wash the beads 3 times with 10 mL of 0.2% SDS/PBS, PBS, and DDW each time. Finally, use 2×0.5 mL DDW to transfer the beads to a low-binding Eppendorf tube and centrifuge, and then remove the supernatant. Trypsin digestion is performed as follows. Pre-mix the following reagents and add to each tube: 200 µL 2M urea/PBS, 2 µL 100 mM CaCl2, and 4 µL trypsin (20 µg reconstituted in 40 µL trypsin buffer). Shake the tube overnight at 37 °C (maximum 12 hours). The beads are then precipitated by short centrifugation, the supernatant and beads are transferred to a small spin column, and the supernatant is collected by centrifugation. The beads were washed with 100 µL PBS and collected in the same tube. The filtrate was acidified with 16 µL formic acid and stored at -20 °C.

The samples were analyzed using LTQ Orbitrap XL ETD (Thermo Fisher Scientific) coupled with NanoLC-2D (Eksigent), and ProLuCID (55) was used to identify proteins, using previously reported parameters (56). Use CImage (57) to calculate the SILAC ratio. The complete SILAC results are available in the MassIVE database under the accession number MSV000084474 (https://doi.org/doi:10.25345/C5P95Q) (58).

The purified human recombinant library was prepared according to the previously reported procedure (18). For labeling experiments, incubate the 1 µM vault with different concentrations of P6 (1, 5, or 10 µM), with or without C12 (1 to 100 µM) on a shaker for half an hour. Labeling, CuAAC chemistry, and in-gel visualization are performed as described above.

( https: //zhanglab.ccmb.med.umich.edu/BSP-SLIM/) (59). Use PyMOL to analyze data and generate numbers.

According to the SI appendix, the joint was synthesized according to the scheme shown in Figure S12. First, 4-oxonon-8-ynoic acid was synthesized as previously reported (52). To 4-oxonon-8-ynoic acid (504 mg, 3 mmol) and 1,2-bis(2-azidoethoxy) (6 g, 10 eq., 30 mmol) in dimethylformamide (DMF) ) In the solution: H2O (9 added: 1, 30 mL), CuCl (94 mg, 0.2 eq., 0.6 mmol) and sodium ascorbate (178 mg, 10.3 eq., 0.9 mmol), and the mixture was stirred at room temperature overnight. The mixture was filtered through a short pad of celite and used without further purification. Crude acid, biotin-ethanolamine (862 mg, 3 mmol), N-hydroxysuccinimide (345 mg, 3 mmol) and 4-dimethylaminopyridine (36 mg, 0.3 mmol) were dissolved in DMF (30 mL). Carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (747 mg, 3.9 mmol) was added portionwise, and the mixture was stirred for 14 hours. Finally, it was concentrated under reduced pressure and purified by reverse-phase high performance liquid chromatography (10%→90% MeCN/0.1% TFA) to obtain the title compound.

The NMR spectrum is shown in the SI appendix, Figure S13; 1H NMR (400 MHz, DMSO): δ 1.21 to 1.40 (m, 2H), 1.40 to 1.69 (m, 4H), 1.79 (quint., J = 7.4 Hz, 2H ), 2.07 (t, J = 7.3 Hz, 2H), 2.54 to 2.63 (m, 3H), 2.69 (t, J = 6.4 Hz, 2H), 2.82 (dd, J = 5 Hz, 12.4 Hz, 1H), 3.06 to 3.13 (m, 1H), 3.26 (quad, J = 5.5 Hz, 2H), 3.37 (m, 3H), 3.52 to 3.58 (m, 6H), 3.81 (t, J = 5 Hz, 3H), 3.98 (t, J = 5.6 Hz, 2H), 4.13 (m, 1H), 4.31 (m, 1H), 4.47 (t, J = 5 Hz, 2H), 6.42 (s, 1H), 6.36 (s, 1H) , 7.82 (s, 1H) and 7.09 (t, J = 5.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 23.6, 24.8, 25.6, 28, 28.5, 28.6, 35.5, 37, 37.9, 41.5, 49.7, 50.4, 55.8, .6905, .616.7. 122.7, 146.7, 163.2, 172.7, 172.8 and 209.1.

To verify that this labeling method is feasible, the cleavable levulinic acid linker was incubated with 50 mM heavy (15N) and light (14N) hydrazine at pH 7.5 overnight, and the reaction was analyzed by LC-MS. Only two main products were detected: dihydropyridazin-3-one and biotinaminoethanol. The desired dihydropyridazine-3-one showed the expected bimodal distribution of heavy cleavage and light cleavage, indicating that two 15N atoms were incorporated in the product, and peaks were detected at 365.31 m/z and 367.28 m/z ( Prediction quality + H: 365.19 and 367.19 m/z represent heavy and light respectively).

The CuAAC reaction proceeded as above, using a cleavable levulinic acid linker (100 µM) to biotinylate the purified 1 µM vault. Enrichment and trypsinization were performed as described above, without a chloroform/methanol precipitation step. Perform data analysis as described above, and then use a custom Python script to identify labeled peptides, which searches for heavy and light labeled peptides with similar intensities (at least 80% similarity) within a 2-minute retention time window. The script can be obtained from https://github.com/barashe/levulinic_linker (60).

C57BL/6 mice were purchased from Jackson Laboratory. MVP-deficient mice and littermate control mice (all mice have a C57BL/6 background) have been described previously (61). BMDM and MEF are prepared using standard protocols. MEF lacking p38α was obtained from p38α loxp/loxp mice and immortalized with retrovirus expressing large T antigen. p38α loxp/loxp mice were kindly provided by Wipinging Jiang, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT. NHBE was obtained from Cambrex and cultivated in accordance with the manufacturer's recommendations. The lung cancer epithelial cell line A549 was purchased from the American Type Culture Collection (ATCC). Normal human CD34+ cord blood cells obtained from StemCell Technologies were cultured according to the manufacturer's recommendations and used to prepare normal human CBM, as previously described (62). MEF and cell lines are maintained in the following growth medium (GM): DMEM (4.5 g/L glucose) supplemented with 10% FBS (HyClone Laboratories, Inc.), L-glutamine, penicillin/streptomycin and optional Amino acid (Invitrogen). BMDM is cultured in 70% GM and 30% L929 conditioned medium. Generally, cells (~60% to 70% confluent) are incubated in the corresponding fresh medium for 12 to 14 hours before stimulation.

The Pseudomonas aeruginosa used in our study was kindly provided by Scott A. Beatson of the University of Queensland in Brisbane, Australia, including wild-type Pseudomonas aeruginosa PAO1 (originally from ATCC) and PAO1 ΔlasI. Bacteria grow on the brain heart infusion (BHI) agar plate, then transfer to the inoculum to cultivate overnight, and then 1/100 dilution into 20 mL BHI broth in a 125 mL baffled shake flask at 37 °C, 250 Incubate at rpm for 4 to 6 hours. Transfer an equal amount of late logarithmic culture or control BHI broth to the PVDF insert and place it in a 24-well plate containing target cells cultured in 0.5 mL of GM as described previously (34) .

Acidify a total of 2 mL bacterial culture with 50 µL HCl, add 10 mL ethyl acetate, and mix the contents vigorously. The layers were separated, the 5 mL ethyl acetate layer was removed, dried over MgSO 4 and concentrated in vacuo. The residue was resuspended in cold methanol and centrifuged to remove the precipitate. The resulting methanol solution was analyzed for C12 content by LC-MS. Reversed-phase LC-MS analysis (Agilent ZORBAX column, 5 µm, 300SB-C8, 4.6 × 50 mm) using MeCN–H2O–0.1% formic acid gradient (from 0 to 1 minute: 5% MeCN, starting from 1 minute) to 9 minutes: gradient from 5% MeCN to 98% MeCN, and from 9 to 11 minutes: 98% MeCN), allowing the quantification of C12 by measuring the following ions: 298 (M + H+), 316 (M + H2O + H+), 320 (M + Na+) and 338 (M + H2O + Na+). The concentration of C12 in the Pseudomonas aeruginosa PAO1 (wild type) sample was approximately 4.3 µM (P <0.001, n = 5 independent experiments). Similar samples from bacteria lacking LasI and from Staphylococcus aureus and Salmonella typhimurium are C12 negative.

Vimentin, HSP70, full-length caspase-3 (casp3FL), caspase-3 cleaved form (casp3cleaved), full-length caspase-8 (casp8FL), caspase-8 cleaved form (casp8cleaved) specific antibodies, full-length caspase-9 (casp9), eIF2α, phospho-eIF2α (p-eIF2α), p38, phospho-p38 (p-p38), MKK3/MKK6, phospo-MKK3 and PARP were purchased from Cell Signaling; anti-actin antibody was from Sigma; anti-MVP Antibodies and anti-PARP4 were from GeneTex. The cell extract was prepared and analyzed by WB as described previously (63).

In all experiments, cells were stimulated with C12 (ranging from 2 to 20 µM), LPS (100 ng/mL), or a combination thereof, as shown in the legend; MEF was stimulated with TNF (40 ng/mL) or KLA ( 50 nM) stimulus, as shown in the text or legend. CBM was stimulated with 10 nM KLA.

ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem, category number 539790) is used to extract proteins based on differences in their subcellular locations. All subcellular grading experiments were performed in accordance with the manufacturer's recommendations.

Use QIAGEN's OneStep RT-PCR kit for XBP1 splicing determination of uXBP1 or sXBP1 mRNA expression. Primer containing XPB1 mRNA splicing sequence (mouse: 5'-GGC CTT GTG GTT GAG AAC CAG GAG-3', 5'-GAA TGC CCA AAA GGA TAT CAG ACT C-3'; human: 5'-GAA CCA GGA GTT AAG ACA GC-3', 5'-AGT CAA TAC CGC CAG AAT CC-3') is used for RT-PCR amplification, the product is passed through 5% polyacrylamide gel in triborate or triacetate -Separate by electrophoresis in EDTA buffer and observe by staining with ethidium bromide.

Since incubating cells with a high concentration of C12 (50 µM or higher) for a long period of time can produce cytotoxic effects (35), in our study, we used a low concentration of C12 (5 to 25 µM for macrophages, fibroblasts And epithelial cells are 10 to 40 µM cells), and the duration of all experiments is limited to 3 hours. Under these conditions, the viability of C12-treated cells was indistinguishable from control cells, which was determined by using an XTT-based toxicology test kit (Sigma).

Metabolic labeling and analysis of sphingosine metabolism were performed according to the method described by Yatomi et al. (64) Modified. BMDM was set in a 30 mm petri dish (about 105 cells per petri dish) and cultured for 2 days. In the presence or absence of 20 µM C12, cells were incubated with 25 nM [3H]-sphingosine (1 µCi). At the specified time, place the petri dish on ice and wash with cold PBS, centrifuge to collect the cells, resuspend in 0.2 mL ice-cold PBS and 1.875 mL containing chloroform/methanol/concentrated HCl (100: 200:1) has been added. The lipid was extracted from the cell suspension by the method of Bligh and Dyer (65). Recover lipids in a small amount of chloroform/methanol (2:1); apply an aliquot (approximately 5,000 cpm per sample) of the lipid fraction to a silica gel 60 TLC plate (Merck) and place the plate in butanol/acetic acid /Water (3:1:1) to expand. Run the appropriate lipid standards on the same TLC plate. The bands were identified by staining the control lipid with primulin and observing under ultraviolet light. After processing the TLC plate with an enhancer (EN3HANCE spray, PerkinElmer Life & Analytical Sciences), use Kodak MS film to perform autoradiography at -80 °C for 20 to 40 hours. In some experiments, radioactive spots were scraped off and counted by liquid scintillation counting. Each autoradiogram shown represents at least three experiments.

The activities of caspase-3, caspase-8 and caspase-9 were measured using the caspase detection kit (R&D System) recommended in the manufacturer's agreement.

The data is depicted in the figure. 1-4 and SI appendix, figure. S1-S11 represent one of three or more experiments. Each chart reflects the typical results of multiple studies. The data is expressed as a relative fold increase compared to the value of untreated or control treated cells (n = 3 to 5).

The complete proteomics and SILAC results are available in the MassIVE database, and the accession number is MSV000084474 (58). The Python script of the peptide tagging strategy using the levulinic acid linker is available on Github: https://github.com/barashe/levulinic_linker (60).

We thank Han Jiahuai for his comments on the manuscript. We would also like to thank Mark Karpasas for his guidance on proteomics analysis. The research was funded by the European Research Council (Initial Grant #240356) to MMM and the US-Israel Science Foundation (Grant #2011360) to MMM. BFCRG thanks the Azrieli Foundation for awarding the Azrieli scholarship.

↵1J.R., RG and NTJ made equal contributions to this work.

Author contributions: research on JR, RG, NTJ, AY, AA, OE, SM, VVK and MMM design; JR, RG, NTJ, LD, AY, AA, OE, SM, VVK and MMM research; JR, RG , RD, LD, AY, MT, BPK, EACW, VAK, LHR, JCM, GFK, TZ, KDJ, RJU, VVK and MMM contributed new reagents/analysis tools; JR, RG, NTJ, AA, OE, EB , SM, BIF, HSO, BFC, TZ, VVK and MMM analysis data; JR, RG, VVK and MMM wrote this paper.

The author declares no competing interests.

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